Long RT-PCR

Paul N Hengen (pnh at fcs260c2.ncifcrf.gov) wrote:
: In article <1994Jun2.181333.24923 at emba.uvm.edu>
:brianf at med.uvm.edu (Brian Foley) writes:
: > .... I found that if I used
: > more plasmid, it was impossible to find optimal conditions. They
: > all worked fine. In fact the conditions that give maximum yield of
: > PCR product when I use just a few copies of plasmid DNA give a poor
: > yield of PCR product if I use 100 nanograms of template DNA to start.
: What happens if you use too much DNA? Do you see the typical large amount of
: EtBr stained material in the well of your agarose gel and smear of DNA like
: that reported by others when trying to amplify large DNA segments with the
: KlenTaqLA-16 enzyme mixture of Barnes? I'm trying to figure out why these
: people can't get it to work. What exactly is the material in the wells?
: Has anyone had this actually work for them?
Yes. If I use too much template, or go too many cycles, or
(sometimes) if I add too much Taq polymerase, I get a lot of DNA in
the well, and a smear of DNA of sizes larger than the expected
product.
If I take samples out of the reaction tube at 5, 10, 12, 15, 17
and 20 cycles durring such an amplification I see first a nice band of
the expected size. Then dimer-sized and trimer-sized bands show up
and the expected sized band begins to disappear. Then these bands
all give way to the smear.
My thought on what is going on is that after the primed-template
pool is exhausted and the Taq goes to work on other things. I have not
tried adding more primers after 10 cycles or doing anything else to
confirm this.
--
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* Brian Foley * If we knew what we were doing *
* Molecular Genetics Dept. * it wouldn't be called research *
* University of Vermont * *
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